The present invention relates to a conjugate comprised of a hydrophobic moiety, a cleavable linkage, and a therapeutic agent. More particularly, the present invention relates to conjugates comprised of a lipid, a cleavable linkage and a drug incorporated into a liposomal formulation. The conjugates are cleavable under mild thiolytic conditions in vivo for release of the drug in an unmodified state.
Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, and in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes. Liposomes having a surface grafted with chains of water-soluble, biocompatible polymer, in particular polyethylene glycol, have become important drug carries. These liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating. The grafted polymer chains shield or mask the liposome, thus minimizing nonspecific interaction by plasma proteins. This in turn slows the rate at which the liposomes are cleared or eliminated in vivo since the Liposome circulate unrecognized by macrophages and other cells of the reticuloendothelial system. Furthermore, due to the so-called enhanced permeability and retention effect, the liposomes tend to accumulate in sites of damaged or expanded vasculature, e.g., tumors, sites of inflammation.
An extended blood circulation time is often desired to allow systemically administered liposomes to reach a target region, cell or site. For example, a blood circulation lifetime of greater than about 12 hours is preferred for liposomal-therapy to a tumor region, as the liposomes must systemically distribute and then extravasate into the tumor region.
One problem associated with liposome-based therapy is retention of drug within the Liposome for a time sufficient for systemic distribution. This problem is of particular concern when long-circulating liposomes, i.e., liposomes with grafted polymer chains, are administered. Relatively few drugs can be efficiently loaded and retained for a long duration and subsequently released.
One approach to improving drug retention is to select lipid bilayer components that render the bilayer less permeable to entrapped drug. However, the lipid bilayer should be sufficiently fluidic such that the drug is released, for example by transport across the lipid bilayer or by lipid vesicle breakdown, at the desired time, e.g., after localization at a target site or sufficient biodistribution.
Another approach to improving drug retention is to covalently attach the drug to a lipid in the liposomal lipid bilayer (Waalkes, et al., Selective Cancer Therap., 6:15-22 (1990); Asai, et al., Biol. Pharm. Bull., 21:766-771 (1998)).
It would be desirable to formulate a liposome composition having a long blood circulation lifetime and capable of retaining an entrapped drug for a desired time, yet able to release the drug on demand. One approach described in the art for achieving these features has been to formulate a liposome from a non-vesicle-forming lipid, such as dioleoylphosphatidylethanolamine (DOPE), and a lipid bilayer stabilizing lipid, such as methoxy-polyethylene glycol-distearoyl phosphatidylethanoLamine (mPEG-DSPE) (Kirpotin, D, et al., FEBS Lett. 388:115-118 (1996)). In this approach, the mPEG is attached to the DSPE via a cleavable linkage. Cleavage of the linkage destabilizes the liposome for a quick release of the liposome contents.
Labile bonds for linking PEG polymer chains to liposomes has been described (U.S. Pat. Nos. 5,013,556, 5,891,468; WO 98/16201). The labile bond in these liposome compositions releases the PEG polymer chains from the liposomes, for example, to expose a surface attached targeting ligand or to trigger fusion of the liposome with a target cell.
To date, however, a means of releasing polymer chains from liposomes under conditions suitable for in vivo use has not been achieved. For example, some releasable linkages require a potent thiolytic agent, such as 1,4-dithiothreitol, to achieve release of the polymer chains. This reducing agent is unacceptable for in vivo use. Another problem with known releasable linkages joining PEG to a liposome lipid is that cleavage of the releasable bond generates an unnatural and undesirable modified lipid. Accordingly, there remains a need in the art for a cleavable linkage that is suitable for in vivo use and which, after cleavage, yields the drug or therapeutic agent in its natural, unmodified form.
In EP 0317957, Senter describes a drug-antibody prodrug, where the antibody is linked to a drug using a disulfide benzyl carbamate or carbonate linker, and reduction of the disulfide bond effects release of the drug. Senter""s teaching is specific to cleavage of a drug-ligand prodrug molecule, under the action of reducing agents such as 1,4-dithiothreitol, glutathione, NADH and NADPH. The behavior of such a linker, when incorporated into a liposome that circulates through the bloodstream, cannot be predicted based on the Senter disclosure. For example, in liposomes, the linker between the polymer and the lipid would be buried or masked in the PEG coating. It is relatively easy to envision release of a single drug-ligand prodrug as in Senter; however, release of the linker when a part of a densely packed barrier as in a liposome surface coating of polymer chains is less predictable.
As noted above, an extended blood circulation time is a desirable feature of PEG-coated liposomes, with blood circulation lifetimes of greater than about 12 hours being preferred for liposomal-therapy to a tumor region. The disclosure of Senter provides no guidance as to the release kinetics of a conjugate incorporated into a liposome under endogenous reducing conditions, such as during blood circulation of the liposome.
Accordingly, it is an object of the invention to provide a liposome composition wherein the drug is retained for a desired period of time for release from the liposome .
It is another object of the invention to provide a conjugate for use in a liposome, wherein the conjugate is comprised of a hydrophobic moiety for anchoring into the lipid bilayer, a linkage cleavable n response to mild thiolytic conditions and a therapeutic agent.
In one aspect, the invention includes a conjugate for use in a liposomal drug-delivery vehicle, the conjugate having the general structural formula: 
wherein L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer, R1 represents a therapeutic drug covalently attached to the dithiobenzyl moiety, and where orientation of the CH2R1 group is selected from the ortho position and the para position.
In one embodiment, the therapeutic drug is covalently attached by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio-carbonate, ether and ester.
In another embodiment, L is selected from the group consisting of cholesterol, a diacylglycerol, a phospholipid and derivatives thereof.
In yet another embodiment, L is a diacylglycerol derivative to yield a conjugate having the general structural formula: 
wherein R2 and R3 are hydrocarbons having between about 8 to about 24 carbon atoms, or in another embodiment, from about 12 to about 22 carbon atoms. In still another embodiment, R2 and R3 are hydrocarbon chains of the same length.
In another embodiment, the drug is selected from the group consisting of mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5-fluorouracil.
In still another embodiment, the therapeutic drug is covalently linked to the dithiobenzyl moiety to form a conjugate having the structure: 
wherein R4 represents a residue of the therapeutic drug.
R4 in one embodiment is a therapeutic drug residue containing a primary or a secondary amine moiety thereby forming a urethane linkage be-ween the dithiobenzyl and the therapeutic drug. In this embodiment, the therapeutic drug can be, for example, mitomycin A, mitomycin C, bleomycin or a polypeptide.
In another embodiment, R4 is a residue of a carboxyl-containing therapeutic drug, which forms an ester linkage between the dithiobenzyl and the therapeutic drug. Examplary drugs in this embodiment include chlorambucil or methotrexate.
In another embodiment, R4 is a therapeutic drug residue containing a hydroxyl moiety thereby to form a carbonate linkage between the dithiobenzyl and the therapeutic drug. Exemplary drugs in this embodiment include fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
In another aspect, the invention includes a liposome composition, comprising liposomes composed of vesicle-forming lipids including from about 1 to about 30 mole percent of a conjugate having the general structural described above. The therapeutic drug is released from the conjugate in vivo in response to a physiologic condition or an artificially induced condition.
In yet another aspect, the invention includes a method for retaining a drug in a liposome, comprising preparing liposomes comprised of a vesicle-forming lipid and of between about 1 to about 30 mole percent of a conjugate described above. The liposomes effectively retain the drug in the liposomes until release from the conjugate in response to a physiologic condition or an artificially induced condition.
These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.